Several homeobox-containing genes related to Drosophila Distal-less (Dll) have been isolated from a wide variety of organisms and have been shown to function as developmental regulators. While in Drosophila only one Dll gene has been described so far, in Vertebrates many components of the Dlx multigenic family have been characterized. This suggests that, during the evolution of the Chordate phylum, the Dlx genes arose from an ancestral Dll/Dlx gene via gene duplication. We have previously reported the isolation of two Dll-related homeoboxes from the protochordate Ciona intestinalis, and described their clustered arrangement (Gene 156 (1995) 253). Here we present the detailed genomic organization and spatial-temporal expression of these two genes, Ci-Dll-A and Ci-Dll-B, and describe the isolation and characterization of another member of the ascidian family of Dll-related genes, which we tentatively named Ci-Dll-C.